Throughout this application various publications are referenced. In some instances, the references are indicated by numerals in parentheses, which numerals refer to a list of citations for the corresponding references that appears at the end of the specification. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains. This application is related to German patent applications 199 08 752.0 and 199 08 766.0, filed on Feb. 19, 1999 and Feb. 17, 2000, respectively, as well as to the corresponding PCT international patent application filed on Feb. 17, 2000. The disclosures of each of these related patent applications is incorporated herein by reference.
The invention relates to compositions and methods for delivery of a molecule into a cell. More particularly, the invention relates to a Vpr polypeptide or fragment thereof conjugated to a molecule, which conjugate is capable of entering the plasma membrane of a cell. The compositions include pharmaceutical and vaccine compositions, and can be used in a variety of methods, including methods to modulate the sensitivity of cells to radiation therapy and to modulate cell proliferation and apoptosis.
Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that encodes the canonical retroviral Gag, Pol, and Env proteins, as well as six regulatory or auxiliary proteins including Tat, Rev, Vpu, Vif, Nef, and Vpr. Although not essential for viral replication in tissue culture, the latter four proteins are highly conserved and likely exert important but less well understood functions in vivo that contribute to viral pathogenesis. Vpr, a xcx9c14-kDa, 96-amino acid protein is conserved among the primate lentiviruses HIV-1, HIV-2, and the simian immunodeficiency virus (SIV), supporting the notion that it serves an important function in the viral life cycle in vivo.
Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral preintegration complex and induces G2 cell-cycle arrest in infected proliferating T lymphocytes. Vpr is highly cytotoxic in pro- and eukaryotic cells and forms homo- and heterologous protein complexes, characteristics that limit the production of recombinant Vpr. Although partial sequences of Vpr have been synthesized for biological (25, 26) and structural studies (32, 33, 34), chemical synthesis of full-length soluble forms of Vpr has proven difficult. Others (27) have reported that Vpr is difficult to synthesize due to its tendency for incomplete coupling, matrix interaction and peptide aggregation. For example, a Vpr peptide derived from the HIV-1BRU sequence has been synthesized, but irreversible aggregation precluded purification of the product (38, 39, 40). The biological activity of such forms of Vpr could not be analyzed in solution. There remains a need for a protocol for high-yield production of synthetic Vpr (sVpr), in which the resultant protein remains both soluble and stable in aqueous solution.
In addition, there remains a need for vehicles capable of delivering molecules into cells. Given that Vpr contains at least two nuclear localization signals (3, 4, 5, 6, 7) and is capable of delivering molecules to the cell nucleus, a vehicle having the nuclear delivery capability of Vpr and also capable of efficiently entering the plasma membrane, preferably without requiring denaturation, is desirable.
The invention meets the above needs and others by providing a composition comprising a Vpr polypeptide conjugated to a therapeutic molecule. In preferred embodiments, the Vpr comprises synthetic Vpr. The synthetic Vpr is preferably stable in aqueous solution, such as the synthetic Vpr produced by the method described herein. The therapeutic molecule can comprise any molecule capable of being conjugated to a Vpr polypeptide, including a second polypeptide, a polynucleotide, and/or a toxin. The toxin can be selected or modified so as to be toxic only to a target cell population. In one embodiment, the toxin or other molecule is modified by conjugation to a regulatory molecule, wherein contact with a target cell affects the regulatory molecule in such a manner as to result in activation of the toxin. For example, the regulatory molecule may be recognized by a protease expressed in the target cell population. Upon contact with a target cell, the protease cleaves the Vpr-toxin conjugate, resulting in activation of the toxin.
The invention additionally provides a method for delivering a molecule into a cell. The method comprises contacting the cell with a conjugate comprising a Vpr polypeptide conjugated to the molecule. The invention further provides a method for modulating the expression of a transgene in a cell. The method comprises contacting the cell with a Vpr polypeptide conjugated to a regulatory molecule. The Vpr:regulatory molecule conjugate, upon contact with the cell, enters the cell and the regulatory molecule modulates the expression of the transgene. In addition, the invention provides a method for killing a target cell population in a subject. The method comprises administering to the subject a Vpr polypeptide:toxin conjugate to the subject. In one embodiment, the toxin is an anti-proliferative agent and the target cell population is cancer cells. The target cell population may be a type of cell that is more susceptible than non-target cells to transduction by Vpr, or it may be a type of cell that is more susceptible than non-target cells to the toxin. In some embodiments, the toxin is further conjugated to a regulatory molecule. Upon contact with a target cell, the regulatory molecule is affected in such a manner as to result in activation of the toxin. In some embodiments, the target cell has been modified to contain a transgene or transgene product whose expression is regulated by the regulatory molecule. In this manner, the invention provides a method for modulating the expression of a transgene in the context of a gene therapy method that results in the killing of cells targeted by a gene therapy protocol.
The invention further provides compositions comprising Vpr, preferably sVpr. Such compositions include pharmaceutical and vaccine compositions, and can be used in a variety of methods. In one embodiment, the invention provides a method for increasing an immune response to an antigen in a subject exposed to the antigen comprising administering Vpr to the subject. The Vpr can be a Vpr polypeptide, and can be administered alone, together with the antigen, or as a Vpr:antigen conjugate. In another embodiment, the invention provides a method for increasing an immune response to HIV in a subject comprising administering a Vpr polypeptide to the subject.
In yet another embodiment, the invention provides a method for increasing the sensitivity of cells to radiation therapy. The invention additionally provides a method for inhibiting cell proliferation. The methods comprise contacting the cells with Vpr. Vpr acts synergistically with radiation to cause G2 arrest in cells, thereby providing a radiosensitizer for use in radiation treatment, such as for treatment of malignancies and other disorders associated with dysregulated cell growth. Vpr can also act alone to inhibit cell proliferation, thereby providing a treatment for hyperproliferative cell disorders, such as malignancies, psoriasis and other disorders associated with dysregulated cell growth. In addition, Vpr can induce apoptosis, and thus can be used in a method of killing cells.